Hematopoietic stem and lymphoid cells engineering from iPSCs:
Nearly all immune cells in our body (blood cells) arise from a specialized cell type in the bone marrow, called Hematopoietic stem cell (HSC). Therefore, any harmful mutation in the HSCs results in genetic diseases of the blood—spanning from cancer to autoimmune diseases. One of the primary goals of this project is to engineer therapeutic HSCs in the laboratory using protein engineering and genetic manipulation techniques. Engineered HSCs are then further differentiated into lymphoid cells (T-cell) using developmental cues and protein engineering methods. Finally, these T-cells are intended to use in the treatment of various genetic diseases.
Brand new protein design:
Proteins are the ‘tiny’ molecular machines in our body that perform everything from powering muscles to transmitting signals in the brain to digesting food. A protein is made from amino acid building blocks--there are twenty such good amino acids--which are strung up and folded into a three-dimensional shape. This shape and the ‘sequence’ of the amino acids in a protein determine its function. A typical size of a protein is about 200 amino acids long, and since there are 20 different amino acids, at least 20200 different proteins are possible in all life forms. However, evolution hasn’t explored this many proteins yet. For instance, humans have about 25,000 unique proteins in their bodies. So there are enormous scopes to build completely brand new proteins (that are not made in nature yet) using evolutionary principles--to cure diseases, fight the climate crisis, and harness energy.
One goal of this project is to build new protein molecules that can self-associate to form larger complexes for a variety of functions. Towards that end, we have built protein assemblies that contain the following number of protein units: 2, 3, 4, 5, 6, 12, and 60. These higher-order proteins were further assembled to form a system-wide network that retained a huge amount of water (called hydrogel). Currently, we are exploring the utility of these protein complexes in the Hematopoietic stem cell differentiation to generate therapeutic T-cells (see the project above).
Intracellular protein delivery:
Due to genetic mutations or other abnormalities, certain protein molecules inside cells lose their functions, leading to many diseases. Therefore, delivering a ‘functional’ protein into the cells to replace the ‘nonfunctional’ one can cure diseases. However, ferrying protein molecules from outside into a cell is an enormous challenge owing to the large sizes of the proteins. We have developed nanotechnological and protein engineering methods that can overcome this challenge. Our method, named the ‘E-tag’ method, was used to deliver a wide range of proteins into a diverse set of mammalian cell systems. Currently, we are interested in this method to deliver protein transcription factors for the generation of Hematopoietic stem cells from patient-derived induced pluripotent stem cells (iPSC).
Gene editing:
CRISPR/Cas9 is the most widely used gene editing tool that has revolutionized the fields of genetics, bioengineering, and medicine. CRISPR/Cas9 is composed of a protein molecule called Cas9 and an RNA molecule called guide-RNA. Owing to the large size of both Cas9 protein and the guide-RNA, it is still a huge challenge to deliver the CRISPR/Cas9-RNP tool to the nucleus of a cell for efficient genome editing.
We used our ‘E-tag’ method (see the section above) to engineer Cas9 protein—we named it Cas9-En—and with the help of engineered nanoparticles, we were able to deliver Cas9-En protein into the cell nucleus with a delivery efficiency of ~90%. Our gene editing platform has been used to engineer therapeutic macrophages and edit mouse spleen cells through in vivo systemic delivery.